We have extended our mechanistic studies of virus-receptor interactions, with focus on two specific enveloped viruses: 1) Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus 8). Following on our previous identification of a novel KSHV receptor protein (xCT, the light chain of the xc- cystine/glutamate exchange transport system), we have generated several reagents for structure-function studies. These include a battery of monoclonal antibodies (Mabs) against glycoprotein H (gH), elicited by immunizing mice with a soluble construct representing the entire extracellular portion of gH. Of the 11 Mabs characterized, all react with the native ectodomain protein captured on ELISA plates, whereas only 4 react with the glycoprotein expressed on the surface of stable gH/gL transfectant cells. The Mabs are being tested for ability to block KSHV glycoprotein-mediated cell fusion and virion entry, using previously described reporter gene systems. Efforts are underway to generate Mabs for the other KSHV glycoproteins involved in fusion/entry, including gB, gL, and K8.1, for which we have generated soluble constructs. We are also pursuing collaborative experiments with investigators at U. Washington to assay KSHV antibodies and antigens in sera and other bodily fluids from KSHV-infected individuals displaying the various related diseases (multicentric Castleman's disease, primary effusion lymphoma, Kaposi's sarcoma). 2) Hepatitis C virus (HCV). Using specialized mammalian producer cells, we have devised a novel system to generate HCV virus-like particles (genotype 1a) in which all viral-encoded components (proteins and RNA) are derived from HCV. We have shown that the system can generate infectious particles carrying full-length HCV genomes that replicate in HCV-permissive target cells. Infection is dependent on HCV glycoprotein interactions with previously identified candidate receptors. We have also adapted the system to generate reporter particles carrying an RNA that when delivered into target cells expressing the appropriate receptors gives rise to a reporter gene signal, independent of the HCV RNA replication machinery. This system provides a means to study HCV entry in diverse target cells, independent of their ability to support HCV replication. Thus diverse target cells can be assessed for their capacity to support HCV entry, an important prerequisite for identifying additional receptor molecules thought to be required for HCV entry. Efforts are underway to test whether this system can be used to generate HCV particles from diverse genotypes, and whether incorporation of fully wild type HCV genomes can give rise to particles that are infectious in vivo. The potential of the HCV particle production system for screening inhibitors of various steps in the virus replication cycle (particle entry, assembly, release, maturation) as well as for HCV vaccine development are under consideration.